Natural killer (NK) cells represent an important component of the immune system. They are a group of white blood cells (lymphocytes) that can directly kill virus-infected cells and tumor cells. NK cells are essential for resistance to many pathogens, such as viruses, bacteria, and parasites. Unlike B and T cells, NK cells do not express antigen-specific receptors, which raises the interesting question of how NK cells can detect infected cells. NK cells exert their function in two ways: by producing cytokines, such as interferon-gamma, and by killing infected cells. Despite the importance of NK cells in the innate immune response to many types of pathogens, it is still unclear what receptors and what signal transduction pathways control their activation. The major goal of this project is to define receptor-ligand interactions that regulate cytotoxicity (lysis of target cells) and cytokine production by NK cells, and to determine the precise contribution of individual receptors to signal transduction in NK cells. Natural killer (NK) cells provide innate control of infected and neoplastic cells. Multiple receptors have been implicated in natural cytotoxicity, but their individual contributions to this process remain unclear. We have studied the activation of resting human NK cells by Drosophila cells expressing ligands for the receptors NKG2D, DNAM-1, 2B4, CD2, and the integrin LFA-1. Each receptor was capable of inducing inside-out signals for LFA-1, including LFA-1 itself, which promoted adhesion. In contrast, none of these receptors alone was able to induce degranulation. Rather, release of cytolytic granules was induced by synergistic activation through co-engagement of receptors, as shown for NKG2D and 2B4. Whereas engagement of NKG2D and 2B4 did not induce strong target cell lysis, collective engagement of LFA-1, NKG2D, and 2B4 defined a minimal requirement for induction of natural cytotoxicity. Remarkably, inside-out signaling induced by each receptor, including LFA-1, was blocked by co-engagement of inhibitory receptor CD94/NKG2A. Inside-out signals induced by the NKG2D and 2B4 combination could overcome the inhibition by CD94/NKG2A. These results detail how activating receptors for natural cytotoxicity co-operate for immunosurveillance and reveal that signals as proximal as inside-out signaling for adhesion are sensitive to inhibition by CD94/NKG2A. By employing Drosophila cells that express ligands of NK cell activation receptors, in the absence of anti-receptor antibodies and exogenous cytokines, we have defined co-engagement of receptors NKG2D, 2B4, and LFA-1 as a minimal requirement for natural cytotoxicity mediated by normal, freshly isolated, resting NK cells. Therefore, our results revealed that target cell recognition by NK cells is a highly dynamic process controlled by the integration of signals from multiple receptors, many of which promote adhesion, some of which synergize to induce degranulation, and all of which are sensitive to inhibitory signaling by receptor CD94/NKG2A. NK cells do not express antigen-specific receptors but are activated by combinations of germ-line encoded receptors. How activation is achieved and controlled during encounter of NK cells with target cells is still unclear. Cytotoxicity of NK cells towards target cells is governed by signals transmitted through multiple receptor-ligand interactions. Activation of freshly isolated, primary NK cells is tightly regulated and requires signaling by the combination of synergistic receptors, which are not activating on their own. The main question addressed here was at what level signals from complementary receptors synergize. Is the requirement for synergy due to the control by distinct receptors of independent steps in the activation pathway, or to a proximal convergence of signals, which may serve to override an early activation threshold? Using co-engagement of receptors NKG2D and 2B4 as a model, we have found that they synergize at a very early activation step, at the level of, or upstream of PLC-gamma activation, which is completely dependent on Vav1. Furthermore, synergy is required to overcome an activation threshold set through inhibition by c-Cbl. These data were obtained using either primary, resting NK cells or an NK cell line that had been rested for a day before use. Stimulation was triggered by crosslinking of receptors with Abs, and was also evaluated in the more physiological context of receptor-ligand interaction between NK cells and target cells. The results have revealed an unsuspected similarity between innate NK cell responses and the priming of naive T cells. For the latter, full activation requires costimulation by CD28 in order to overcome inhibition by Cbl-b, whereas NK cells require a synergy of co-activation receptors to overcome inhibition by c-Cbl. Cytotoxic lymphocytes, which include NK cells, kill target cells by releasing the content of secretory lysosomes at the site of contact with target cells, which has been called the cytotoxic immune synapse. To understand the dynamics and control of cytotoxic immune synapses we imaged human primary NK cells on lipid bilayers carrying ligands of activation receptors. These were the first live images of cytotoxic immune synapses during degranulation, and of NK cell immune synapses. Formation of an organized synapse was dependent on the presence of the beta2 integrin ligand ICAM-1. Ligands of coactivation receptors 2B4 and NKG2D segregated into central and peripheral regions, respectively. Lysosomal protein LAMP-1 that was exocytosed during degranulation accumulated in a large and spatially stable cluster. A striking feature of NK cell cytotoxic immune synapses formed in the presence of ICAM-1 is that LAMP-1 delivered to the cell surface did not escape and diffuse over the plasma membrane. Instead, LAMP-1 accumulated in the central cluster, which overlapped with a site of membrane internalization. Imaging of NK cells at the earliest stages of degranulation suggested that ICAM-1 promotes LAMP-1 exocytosis at the center of the synapse, rather than LAMP-1 retrieval from the periphery. Furthermore, live imaging of lysosomal compartments showed that they reached the plasma membrane at focal points directly adjacent to centrally accumulated LAMP-1. This stable region of membrane dynamics is the defining feature of cytotoxic immune synapses, as it could occur even in the absence of an organized distribution of receptors at the synapse. Therefore, our work has shown that cytotoxic immune synapses include a central region of bidirectional vesicular traffic, which is controlled by integrin signaling.